Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory CNS demyelinating disease that serves as a model for human multiple sclerosis (MS). A relapsing-remitting form of EAE (R-EAE) is induced in SJL/J mice following active immunization with either the major encephalitogenic determinants of proteolipid protein (PLP139-151) or myelin basic protein (MBP84-104) or by the adoptive transfer of peptide-specific T cells. Our productive studies during the previous funding period proved that pathologic disease progression (relapses) in these peptide-induced R-EAE models is due primarily to the recruitment of T cell responses against non-crossreactive endogenous myelin epitopes on the same or different myelin proteins (intramolecular or intermolecular epitope spreading). Since numerous human autoimmune diseases (e.g., MS, rheumatoid arthritis, SLE) display relapsing-remitting or chronic-progressive courses, delineating the cellular and molecular mechanisms of epitope spreading has important implications for understanding the pathogenesis of these diseases and for the potential application of specific forms of immunotherapy for their treatment. We will test the hypothesis that priming of T cells specific for endogenous epitopes occurs primarily in the CNS itself. Specific Aim 1 will use a variety of approaches to precisely quantitate the temporal appearance of Th1/Th2 cells in peripheral lymphoid organs, peripheral blood and the CNS specific for a panel of encephalitogenic myelin epitopes (using ELISPOT analysis, intracellular cytokine staining, and MHC class II peptide-Ig dimers), their T cell receptor (TCR) repertoire (using immunoscope), and their encephalitogenic potential. Specific Aim 2 will utilize a transgenic T cell transfer system to analyze the temporal appearance and anatomic location of activation of myelin-specific T cells stimulated by endogenous myelin epitopes released during acute clinical disease. Specific Aim 3 will delineate the differential expression of molecules required for antigen presentation (e.g., MHC class II processing machinery, cathepsins, and costimulatory molecules) in and determine the relative functional ability of peripheral (dendritic cells, macrophages, and B cells) and CNS-resident APC populations (astrocytes, microglia, infiltrating macrophages, and cerebrovascular endothelial cells) derived from primary cultures and isolated from mice with ongoing EAE to process and present encephalitogenic myelin epitopes on PLP, MBP, MOG to a panel of myelin epitope-specific naive T cells and activated Th1/Th2 clones, These studies should enhance our understanding of the mechanisms of induction of epitope spreading and the effector role of these responses in the chronic pathogenesis of CNS and other autoimmune diseases and provide vital information for the design of immunoregulatory strategies for their treatment.